S274: A NOVEL SUBTYPE OF ANEMIA CAUSED BY MUTATIONS IN TFRC GENE
Authors: S. Colucci, V. Venturi, F. Nicole, D. Jové Solavera, M. Zimon, P. Richter-Pechanska, G. Hernandez, S. Unal, F. Gumruk, A. Diaz-Conradi, L. Romero-Cortadellas, X. Ferrer-Cortès, M. Olivella, M. Erlacher, C. Niemeyer, T. Wiesel, R. Pepperkok, M. D. Fleming, A. E. Kulozik, M. Sanchez, M. U. Muckenthaler
Journal: HemaSphere (2022)

Background: Anemia affects ~25% of the world population and is frequently caused by iron deficiency as a consequence of malnutrition or inflammation. Only in rare cases anemia is due to mutations in genes responsible for balancing iron homeostasis. Among these, loss of function variants of MT2 cause iron-refractory iron-deficiency anemia (IRIDA); a disease characterized by inappropriately high levels of hepcidin. Hepcidin is a hepatic hormone that limits dietary iron uptake and iron release from intracellular stores and thus renders IRIDA patients resistant to iron therapies. In mice, a form of anemia resistant to iron supplementation is further caused by mutations in Transferrin Receptor 1 (TfR1; Tfrc gene). TfR1 internalizes iron-bound transferrin, a process fundamental for erythropoiesis. However, up to date, the sole human report describing a TfR1 mutation (p.Tyr20His) showed that it impairs the immunological compartment rather than erythroblast maturation. Here, we report pediatric patients with previously undescribed mutations in TFRC, diagnosed with microcytic hypochromic anemia, partially resistant to iron therapies. Aims: Identification of novel disease alleles that cause anemia. Methods: Whole exome sequencing (WES) of DNA of patients with anemia. Functional characterization of novel mutations identified in TFRC gene was assessed in HeLa cells overexpressing TfR1 wild-type and mutated by western blotting and wide-field microscopy. Results: A 4-year old boy manifested symptoms of anemia and the analysis of hematological parameters revealed low levels of hemoglobin, MCV and MCH. Plasma iron, transferrin saturation and hepcidin were within physiological ranges. Unexpectedly, soluble TfR1 (sTfR1) levels were almost undetectable and the bone marrow smear revealed increased erythropoiesis and iron deficiency in this compartment. The condition of hypochromic microcytic anemia was persistent over years and could not be corrected by oral iron therapy. WES analysis identified a TFRC_c.941C>T homozygous mutation, encoding for TfR1P314L. Relatives carrying the same mutation in heterozygosity were not anemic. This indicates that TFRC_c.941C>T mutation causes a recessive form of anemia. Overexpression of a construct expressing the TfR1P314L mutant in HeLa cells showed a reduction in total and secreted TfR1, thus reflecting upon the observation of very low sTfR1 levels in the blood of the patient. We further show that lower TfR1 levels are likely due to a faster degradation rate of TfR1P314L compared to TfR1WT. In addition, the TfR1P314L mutated protein showed impaired internalization of fluorescently labeled transferrin, most likely explaining the reduced iron levels observed in the bone marrow. In addition to this index patient, 3 additional children of 2 independent families were identified with TFRC mutations and hypochromic microcytic anemia. The genetic analysis detected a TFRC_c.967C>G heterozygous mutation encoding for TfR1P323A and a TFRC_c.934G>A homozygous mutation, encoding for TfR1G312R. All three TFRC mutations are highly conserved across species and are located in the same extracellular loop of TfR1. Summary/Conclusion: We identified a novel subtype of juvenile hereditary hypochromic microcytic anemia that is partially resistant to iron therapies and caused by missense mutations in the TFRC gene. Reduced endocytosis of iron-bound transferrin may contribute to insufficient iron supply for erythropoiesis and resistance to iron supplementation. In the clinical practice, our findings suggest to include TFRC sequencing analysis for patients with unexplained anemia.